Bacterial Communities Associated with the Pine Wilt Disease Vector Monochamus Alternatus (Coleoptera: Cerambycidae) During Different Larval Instars

Journal of Insect Science, (2017)17(6): 115; 1–7 doi: 10.1093/jisesa/iex089 Research Article

Bacterial Communities Associated With the Pine Wilt Disease Vector Monochamus alternatus (Coleoptera: Cerambycidae) During Different Larval Instars

Xia Hu,1 Ming Li,1 Kenneth F. Raffa,2 Qiaoyu Luo,1 Huijing Fu,1 Songqing Wu,1 Guanghong Liang,1 Rong Wang,1 and Feiping Zhang1,3

1College of Forestry, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China, 2Department of Entomology, University of Wisconsin-Madison, 345 Russell Labs 1630 Linden Dr., Madison, WI 53706, and 3Corresponding author, e-mail: [email protected]

Subject Editor: Campbell Mary and Lancette Josh

Received 14 June 2017; Editorial decision 20 September 2017

Abstract We investigated the influence of larval instar on the structure of the gut bacterial community in the Japanese pine sawyer, Monochamus alternatus (Hope; Coleoptera: Cerambycidae). The diversity of the gut bacterial community in early, phloem-feeding larvae is significantly higher than in later, wood-feeding larvae. Many of these associates were assigned into a few taxonomic groups, of which Enterobacteriaceae was the most abundant order. The predominant bacterial genus varied during the five instars of larval development.Erwinia was the most abundant genus in the first and fifth instars,Enterobacter was predominant in the third and fourth instars, and the predominant genus in the second instars was in the Enterobacteriaceae (genus unclassified). Actinobacteria were reported in association with M. alternatus for the first time in this study. Cellulomonadaceae (Actinobacteria) was the second most abundant family in the first instar larvae (10.6%). These data contribute to our understanding of the relationships among gut bacteria and M. alternatus, and could aid the development of new pest control strategies.

Key words: gut bacteria, pyrosequencing, long-horned beetle, Enterobacteriaceae

Larvae of long-horned beetles (Cerambycidae) are xylophagous, and Iwasaki 1972, Teale et al. 2011). This insect-transmitted patho- which feed in subcortical tissues of healthy, dead, or decaying woody gen has caused significant losses of pines in Japan, Korea, China, and plants (Haack and Slansky 1987, Grünwald et al. 2010). Larval Portugal (Rodrigues 2009, Chen et al. 2013, Alves et al. 2016, Van development occurs entirely within the host, requires at least several Nguyen et al. 2017). One of the major strategies to manage the nem- months, and can kill trees (Allison et al. 2004). Bacterial communi- atode is to reduce between-tree transport by controlling M. alterna- ties associated with subcortically feeding beetles are known to play tus. Owing to the importance of M. alternatus, various aspects of its important roles in facilitating larvae in surviving and developing physiology and genetics have been studied, such as its pheromones within their host plants (Douglas 2009; Scully et al. 2013, 2014; (Teale et al. 2011), transcriptome (Wu et al. 2016), pathogens (Ma Alves et al. 2016). Bacterial communities are reported to contribute et al. 2009), symbiotic fungi (Maehara et al. 2005) and tracheal to their host beetles’ reproductive success, community interactions bacteria (Alves et al. 2016). However, symbiotic intestinal bacterial and niche diversification (Cardoza et al. 2006, Scott et al. 2008, communities are not well known for M. alternatus. Douglas 2009, Morales-Jiménez et al. 2013). Bacteria can contribute Like other Monochamus spp., M. alternatus feed on different to the nutrition of phloeophagous and xylophagous larvae, which sections of the wood during different larval stars. After hatching, rely on a nutrient-poor food source, by exploiting nitrogen and car- early larvae feed first on phloem under bark. Later larvae feed in the bon compounds in woody substrates and providing nutritional sup- xylem, including sapwood and heartwood, and form long, irregular plements that are absent from the substrate, such as amino acids mines (Yanega 1996). Thus, their food source changes substantially and essential vitamins (Dillon and Dillon 2004, Geib et al. 2008, during development, and previous studies have not yet categorized Morales-Jiménez et al. 2009, Berasategui et al. 2017). how corresponding gut communities respond (Park et al. 2007, Ma The Japanese pine sawyer, Monochamus alternatus (Hope; et al. 2009, Scully et al. 2014, Alves et al. 2016). Coleoptera: Cerambycidae), is the most important vector in Asia A deeper understanding of the structure of the microbiome of the for long-distance transport of the pine wood nematode (PWN), insect vector is required, and may contribute to the development of Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle 1970, the new approaches to managing PWD. To better understand how do invasive pathogen that causes pine wilt disease (PWD), (Morimoto symbiotic intestinal bacterial communities relate to larval feeding

© The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ 1 licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 2 Journal of Insect Science, 2017, Vol. 17, No. 6 stage, we used a metagenomics approach to investigate the gut-asso- platform (San Diego, CA) for sequencing according to the stand- ciated bacteria diversity and community structures. ard protocols at Majorbio Bio-Pharm Technology, Shanghai, China. Raw fastq files were then demultiplexed and quality-filtered by using Materials and Methods QIIME (version 1.17). After pyrosequencing, the quality of the raw Miseq sequenc- Insect Collection and Dissection ing reads was checked with FastQC (Margulies et al. 2005, Andrews Larvae of M. alternatus in different instars were removed from 2014). Raw reads were quality screened by using an average minimum recently attacked Pinus massoniana (Lamb; Pinales: Pinaceae). quality score of 20. Barcodes and primers sequence were trimmed by Larvae in the first and second instar were collected from phloem and using the Trimmomatic. After quality control and barcode assignment, third, fourth, and fifth instar larvae were collected from sapwood operational taxonomic units (OTUs) were clustered with 97% simi- and heartwood. Larvae were placed on ice, and then transported to larity cutoff using Usearch (version 7.1) and chimeric sequences were the laboratory in sterile vials containing sterile moist paper. Sampling identified and removed using UCHIME. Mothur (http://www.mothur. was performed in the town of Guan Tou, Lianjiang county in Fujian org/) was used to sort sequences exactly matching the specific barcodes Province (N 26.15046°; E 119.59261°) in August 2015. All larvae into different samples. Then, Sickle tool (https://github.com/najoshi/ were manually removed directly from galleries using fine forceps. sickle) was used to perform the quality filtering to remove the reads with Instars first through fifth were separated according to the width their average quality score <50 or with any unknown bases. Then, the reads head capsules (Liu et al. 2008) (Table 1). Three larvae in each instar were assembled by Mothur command ‘make.contigs’ with the criteria of were prepared for 16S rRNA analysis, for a total of 15 samples. ‘maxambig = 0’, ‘maxhomop = 8’, and ‘minoverlap = 10’. The quality-fil- The larvae were surface sterilized with 70% ethanol for 1 min, tered reads were then processed by Mothur with commands ‘trim.seq’, and then rinsed twice with sterile water. After placing in 10 mM ster- ‘pre.cluster’, and ‘chimera.uchime’ to remove chimera and sequencing ilized phosphate-buffered saline (138 mM NaCl and 2.7 mM KCl, noise (Guo et al. 2015). Taxonomic classification of each sample was pH 7.4), the larvae were dissected under a stereomicroscope using individually conducted using Ribosomal Database Project (RDP) (http:// insect pins to obtain mid-guts and hindguts. One gut from each larva rdp.cme.msu.edu/) Classifier (version 2.6) with a confidence threshold was transferred to a 1.5-ml microcentrifuge tube with 500 ml of tris- of 50% (DeSantis et al. 2006, Wang et al. 2007, Cole et al. 2009, Quast EDTA (10 mM tris-HCl [pH 8.0], 1 mM EDTA) separately and then et al. 2013). ‘Aligner’ and ‘Complete Linkage Clustering’ were applied to homogenized several times with a plastic pestle, followed by vortex- calculate richness and diversity indices including OTUs, Shannon Index, ing for 3 min at the speed of 2500 r/min. The homogenate was cen- and Chaos index (Schloss et al. 2011). Sequences were rarefied to the trifuged at 4000 r/min for 15 s to separate the microbial cells from lowest number of reads in the samples using QIIME script single_rar- the gut wall tissues and undigested food (Hu et al. 2013). The super- efaction.py. before statistical analysis. Rarefaction curve methodology natant (containing bacteria) was transferred to new tubes for DNA was used to estimate the relationship between the expected OTU rich- extraction. All procedures were completed in a sterile environment. ness and sampling depth (Colwell et al. 2004).

DNA Extraction and PCR Amplification Statistical Analysis DNA was extracted from the samples using QIAamp Fast DNA Principal component analysis (PCA) was performed using the vegan Stool Mini Kit (Qiagen, Germany). Successful DNA isolation was package for R (version 2.1) (Wang et al. 2012). Potential significant confirmed by agarose gel electrophoresis. DNA concentration was differences were analyzed by t-test, analysis of variance (ANOVA) and assessed by a Nanodrop (Thermo Scientific) and quality was deter- chi-square in SPSS version 18.0. (SPSS Inc.: Chicago, IL). Normality mined by agarose gel electrophoresis. The V3-V4 regions of the bac- of the data was evaluated with the Kolmogorov Smirnov test. teria 16S ribosomal RNA gene were amplified by PCR using the following primers: 338F 5ʹ-barcode-ACTCCTACGGGAGGCAG- CA-3ʹ and 806R 5ʹ-GGACTACHVGGGTWTCTAAT-3ʹ (barcode Results is an 8-base sequence unique to each sample). The PCR products Pyrosequencing were then extracted from 2% agarose gels, and further purified by A total of 13 samples were successfully sequenced, which excluded using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences) the data from one first instar and one third instar. The raw data set and quantified by QuantiFluor-ST (Promega). of all 13 samples contained 408,318 reads. After stringent quality assessment and data filtering, 79.2% high-quality reads were avail- High-Throughput Pyrosequencing and Bacterial able for analysis. The average length of valid sequences was 448 bp. Community Analysis A total of 159,106 valid sequences (123 Mb in total) obtained The purified DNA amplicons were then added with Illumina adapt- from genomic DNA harvested from these gut-associated bacteria ers by ligation (TruSeq DNA LT Sample Prep Kit), and the adapter-li- have been deposited into GenBank database (accession number: gated DNA fragments were further amplified on an Illumina MiSeq SRX2251637-SRX2251649).

Table 1. Mean width of larval heads and pronotum (±SE) of Monochamus alternatus, n = 3 samples per instar

Width of samples (mm) Instar

1st 2nd 3rd 4th 5th

Larval head 1.08 ± 0.04 1.66 ± 0.05 2.68 ± 0.03 3.14 ± 0.04 3.49 ± 0.01 Larval pronotum 1.57 ± 0.12 2.03 ± 0.09 3.84 ± 0.04 4.64 ± 0.18 5.32 ± 0.06 Journal of Insect Science, 2017, Vol. 17, No. 6 3

Bacterial Diversity Analysis and OTUs Bacteroidetes comprised 2.0%. In fifth instar larvae, Proteobacteria To determine community richness and diversity, the Shannon index accounted for 97.3% (Fig. 2). of diversity (H′) and Simpson index (S) were determined for all sam- Enterobacteriaceae (Proteobacteria) was the most abun- ples (Table 2, Supplementary Table S1). The value of H′ ranged from dant family in all samples, representing 86.1%, 94.5%, 97.7%, 0.09 to 1.48, and the value of S ranged from 0.29 to 0.97. The H′ 86.6%, and 91.9% from first to fifth instar larvae, respec- index showed that there were no significant differences among dif- tively. The Enterobacteriaceae present included Enterobacter, ferent stages (Kruskal-Wallis tests, χ2 = 5.5238, df = 4, P = 0.238). Erwinia and Enterobacteriaceae-like genera shown in the Fig. 3. The S index, which gives more weight to dominant species, showed Cellulomonadaceae (Actinobacteria) was the second most abun- that significant differences among different stages (Kruskal-Wallis dant family in the first instar larvae (10.6%). At the genus level, tests, χ2 = 10.341, df = 4, P = 0.035). The diversity of bacteria in all samples share some taxa. Enterobacter (Enterobacteriaceae, early larvae (first, second instars) was significantly higher than γ-Proteobacteria), Erwinia (Enterobacteriaceae, γ-Proteobacteria), that of the later larvae (third, fourth, fifth instars) (P < 0.05), with Gordonia (Moraxellaceae, Actinobacteria), and Chryseobacterium Student’s t-tests for S index (F = 27.3, df = 11, P < 0.001) and H′ (Flavobacteriaceae, Bacteroidetes) were each present in at least one index (F = 5.5, df = 11, P = 0.038). larval gut of all M. alternatus instars (Fig. 3). OTUs were identified at genetic distances of 0.03 (species level), Combining the relative abundance of reads and the results of 0.05 (genus level) and 0.2 (phylum level) by using quality sequences the taxonomic-based analysis, Erwinia is the most abundant bacterial genus in first (58.3 ± 3.7%) and fifth (82.0 ± 16.9%) instar with a read length of ≥50 bp per sample. A total of 75 OTUs0.03 were obtained from the 13 samples (Supplementary Table S1, S2, larval guts. Enterobacter is the predominant bacterial genus in third Fig. S1). The number of OTUs ranged from 9 to 44 clusters per sam- (97.1 ± 2.3%) and second (49.2 ± 14.2%) instar guts, and of varia- ple. There were no significant differences among different instars ble relative prevalence in first (9.8 ± 2.5%), second (31.7 ± 20.1%), (Kruskal-Wallis tests, χ2 = 5.329, df = 4, P = 0.255), and no signif- and fifth (4.4 ± 7.4%) instar guts. Enterobacteriaceae are predom- icant differences between early versus later larvae (t-tests F = 0.6, inant in the second instar guts (60.7 ± 9.5%). Its frequencies in P = 0.454) in the number of OTUs. There were 145,085 reads other stages were: first: 18.0 ± 2.7%, fourth: 30.5 ± 9.6%, fifth: belonging to six clusters (OTU3, OTU9, OTU28, OTU40, OTU55, 5.0 ± 8.3%. Genera belonging to Cellulomonadaceae comprised the and OTU62), which accounted for 91.2% of the total 159,106 reads third most abundant bacteria in the first (10.6 ± 4.5%) instar guts. (Supplementary Table S2). According to the analysis of the shared The genus Lactococcus (Streptococcaceae, Firmicutes) was the third and unique OTUs and reads between the different instars (Fig. 1), abundant bacteria in fourth (13.0 ± 19.8%) instar guts. the gut associated bacterial communities hosted by M. alternatus PCA indicated potentially correlated variables among bacterial early larvae displayed some commonalities with later larvae. A total distributions. The first two principal components (PCs) explained of 54 OTUs and 20,758 reads were shared between early larvae 67.2% of the variance of the bacterial communities (Fig. 4). PCA (including first and second instars) and later larvae (including third, showed that samples obtained in different instars clustered into five fourth, fifth instars) (Fig. 1c and 1f). separate groups, while, B3 (second instar) and E1 (fifth instar) were clustered with D (fourth instar). This analysis indicated that the gut Taxonomic Assignment of Bacterial Symbionts and bacterial communities from larvae within the same instar tended to Dominant Taxa be close. After taxonomic-based analysis, all OTUs were assigned to four different phyla: Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria. At the phylum level, the composition of the bac- Discussion terial community structure was similar across all five instars This study provides new insight into the gut bacterial diversity of (Fig. 2, Supplementary Fig. S2). Proteobacteria was the dominant M. alternatus among different larval instars by high-throughput phylum, representing on average 93.1% of the reads in each sam- pyrosequencing. Overall, the diversity of gut-associated bacteria in ple, followed by Firmicutes (3.2%), Bacteroidetes (2.6%), and early larvae was higher than in later larvae, which might be related Actinobacteria (1.0%) (Fig. 2, Supplementary Table S3). In first to dietary differences between them. Proteobacteria was the most instar larvae, Proteobacteria comprised 86.9% and Actinobacteria dominant phylum, representing on average 93.1% of the reads comprised 12.4%. Proteobacteria accounted for 97.7% in second in each M. alternatus sample. In particular, genera in the family instar and 98.6% in third instar larvae. In the fourth instar larvae, Enterobacteriaceae (γ-Proteobacteria) represent a major fraction, Proteobacteria comprised 85.0%, Firmicutes comprised 13.0% and and occurred in the guts of all larvae sampled. γ-Proteobacteria

Table 2. Total number of reads obtained by pyrosequencing for samples of each instar, and the respective indexes of diversity including the number of OTUs, Shannon-Wiener index (H′) and Simpson index (S)

Larval stage Reads Similarity threshold (0.97)

OTU Ace Chao1 Coverage H′ S

1st 10858 24 36 29 0.999355 1.24 ± 0.01 0.40 ± 0.02 2nd 12525 22 37 27 0.999491 0.92 ± 0.10 0.50 ± 0.01 3rd 11406 18 23 21 0.999606 0.19 ± 0.07 0.94 ± 0.02 4th 11924 19 30 24 0.999406 1.26 ± 0.07 0.33 ± 0.01 5th 13744 36 38 37 0.999661 0.74 ± 0.19 0.71 ± 0.08

Data of Reads, OTU, ace, chao and coverage are means; data of H′ and S are means ± SE, n = 2 (1st and 3rd), and n = 3 (2nd, 4th, 5th). 4 Journal of Insect Science, 2017, Vol. 17, No. 6

Fig. 1. Venn diagrams representing the number of shared and unique OTUs and reads. (A) OTUs between first and second instar larvae; (B) OTUs among third, fourth, fifth instar larvae; (C) OTUs between early larvae and later larvae; (D) reads between first and second instar larvae; (E) reads among third, fourth, fifth instar larvae; (F) reads between early larvae and later larvae.

could be detected in one of these isolates (Park et al. 2007). Likewise, in our other culture-dependent research, several cellulolytic bacteria belonging to Proteobacteria, Firmicutes, and Bacteroidetes were also found in the guts of M. alternatus larvae. It was confirmed that gut associated bacteria could help host insects degrade wood fabric. Like other wood-boring beetles, M. alternatus was associated with a core group of microbiota, which seem likely to influence host success (Hu et al. 2013, Mason et al. 2016). Erwinia and Enterobacter are consistently found in a variety of insect guts, including Diptera, Lepidoptera, Homoptera, Coleoptera (Harada et al. 1997, Watanabe and Sato 1998, Basset et al. 2003, Hu et al. 2013, Aylward et al. 2014, Mason et al. 2016). Interestingly, Enterobacter is also associated with PWN carried by M. alternatus in China (Han et al. 2003). This supports the hypothesis that PWN can harbor bacteria from the insect. These representatives of the Enterobacteriaceae might benefit xylophagous hosts because Fig. 2. Relative abundance of the predominant bacterial phylum from each insect sample (more than 0.1% of the total number of reads). Data shown of their ability to hydrolyze many polysaccharides (Scully et al. are means. 2013). A few species can persist in the gut’s harsh environment, which consists of digestive enzymes, high redox potential and high (87.9%) was also reported to associate with Monochamus gallopro- ionic strength (Vallet-Gely et al. 2008). Erwinia are likely to have vincialis (Olivier; Coleoptera: Cerambycidae), an important vector been acquired during larval feeding, based on documentation of of PWN in Europe (Sousa et al. 2001, Naves et al. 2007, Vicente this pathway in Drosophila melanogaster Meigen and Frankliniella et al. 2013). The predominance of Enterobacteriaceae in the gut bac- occidentalis Pergande (De Vries et al. 2001, 2004; Basset et al. terial community of M. alternatus is in agreement with reports from 2003). Erwinia produce a set of depolymerizing enzymes, such as other phytophagous insects from several feeding guilds (Broderick pectinases, cellulases, proteases, phospholipases and xylanases that et al. 2004, Delalibera et al. 2005, Schloss et al. 2006, Hu et al. can degrade plant cell wall components, and include some phyto- 2016). Enterobacteriaceae were also found to be the predominant pathogenic species (Barras et al. 1994). Interestingly, Erwinia was family (52.2%) in the bacterial communities colonizing the trachea also the dominant genus, and its relative abundance changed during of M. galloprovincialis and M. alternatus (Alves et al. 2016). In the different development stages, in the guts of thrips (De Vries et al. abdomen of M. galloprovincialis, the most abundant genus was 2001). Thrips benefitted from gutErwinia when they fed on a diet Serratia (95%) (Enterobacteriaceae) (Vicente et al. 2013), whereas of only leaves, but experienced negative effects of Erwinia when no Serratia was detected in M. alternatus gut in this work. The gen- they fed on a mixed diet of leaves with pollen (De Vries et al. 2004). era composed bacterial community might varied among different This suggests that Erwinia might represent a diet-dependent switch Monochamus spp. In the only culture-dependent study of gut-associ- from mutualism to parasitism in some host insects. The prevalence ated bacteria of Monochamus spp., 14 of 16 strains were identified of Erwinia in all five instars might also be related to digestion of as γ-Proteobacteria in the gut of M. alternatus, with the other two lignocellulose. The relative abundances of Erwinia among different strains belonging to Firmicutes (Park et al. 2007). Xylanase activity instar larvae might be influenced by differences in feeding substrates. Journal of Insect Science, 2017, Vol. 17, No. 6 5

Fig. 3. Relative abundance of the predominant bacterial genus for each insect sample (only genera with more than 0.1% relative abundance were shown).

bacteria may be sources of enzyme that contribute to nutrition of the host insect. The highest levels of Enterobacter were found in the third and fourth instar communities, with a high abundance also showing in other instars. Enterobacter was also predominant in the larval gut of long-horned beetle Rhagium inquisitor L. (Coleoptera: Cerambycidae) (Grünwald et al. 2010). These results could potentially lead to improved protection of pine from the M. alternatus–B. xylophilus complex. Enterobacter gergoviae, a gut bacterium of the pink bollworm (Pectinophora gossypiella Saunders), was exploited as a biopesticide vector by transforming it to express Cyt1A, an insecticidal protein lethal to mosquitoes and black fly larvae (Kuzina et al. 2002). Also, the gut bacterium Enterobacter cloacae was used to control the mulberry pyralid (Glyphodes pyloalis Walker; Lepidoptera: Crambidae) by transforming the ice nucleation gene to increase the supercooling point of G. pyloalis, thus causing increased mortality (Watanabe et al. 2000). Detailed knowledge about the functions of these gut bacteria and their modes of transmission are necessary before similar strategies could be successfully devised and implemented against

Fig. 4. Principal component analysis (PCA) showing the potentially correlated invasive longhorned beetles or their phytopathogenic symbionts. variables of the bacterial distribution pattern in each Monochamus alternatus The results enlarged our understanding of the relationships among sample: A1, A2 represent first instar samples; B1, B2, B3 represent second gut bacteria and M. alternatus, and could encourage the develop- instar samples; C1, C2 represent third instar samples; D1, D2, D3 represent ment of new pest control strategies in the future. fourth instar samples; E1, E2, E3 represent fifth instar samples.

Supplementary Material Cellulomonadaceae (Actinobacteria), another major component in first instar larval gut, are known to produce a large vari- Supplementary material can be found at Journal of Insect Science ety of hydrolytic starch, xylan and cellulose-degrading enzymes online. (Stackebrandt et al. 2006). Actinobacteria were reported for the first time in association with M. alternatus in this study. Lactococcus (Firmicutes) is the third abundant genus of fourth instar larval gut Acknowledgments bacteria. In nature, L. lactis occupies a niche consisting of plant or We acknowledge the Special Fund for Forestry Research in the Public Interest of animal surfaces and the animal gastrointestinal tract. It is believed China (Grant No. 201304401), Natural Science Foundation of Fujian Province to be dormant on the plant surfaces and to multiply in the intesti- (Grant No. 2016J05064), the Special Fund for Key Construction Project of nal tract after feeding (Bolotin et al. 2001). All these dominant gut Fujian Agriculture and Forestry University (Grant No. 6112C035005). 6 Journal of Insect Science, 2017, Vol. 17, No. 6

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